Clock Neurons Research Articles

Drosophila CrebB is a Substrate of the Nonsense-Mediated mRNA Decay Pathway that Sustains Circadian Behaviors.

Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.

Differential regulation of the Drosophila sleep homeostat by circadian and arousal inputs.

One output arm of the sleep homeostat in Drosophila appears to be a group of neurons with projections to the dorsal fan-shaped body (dFB neurons) of the central complex in the brain. However, neurons that regulate the sleep homeostat remain poorly understood. Using neurogenetic approaches combined with Ca2+ imaging, we characterized synaptic connections between dFB neurons and distinct sets of upstream sleep-regulatory neurons. One group of the sleep-promoting upstream neurons is a set of circadian pacemaker neurons that activates dFB neurons via direct glutaminergic excitatory synaptic connections. Opposing this population, a group of arousal-promoting neurons downregulates dFB axonal output with dopamine. Co-activating these two inputs leads to frequent shifts between sleep and wake states. We also show that dFB neurons release the neurotransmitter GABA and inhibit octopaminergic arousal neurons. We propose that dFB neurons integrate synaptic inputs from distinct sets of upstream sleep-promoting circadian clock neurons, and arousal neurons.

Age-dependent changes in clock neuron structural plasticity and excitability are associated with a decrease in circadian output behavior and sleep

Aging has significant effects on circadian behavior across a wide variety of species, but the underlying mechanisms are poorly understood. Previous work has demonstrated the age-dependent decline in behavioral output in the model organism Drosophila. We demonstrate that this age-dependent decline in circadian output is combined with changes in daily activity of Drosophila. Aging also has a large impact on sleep behavior, significantly increasing sleep duration while reducing latency. We used electrophysiology to record from large ventral lateral neurons of the Drosophila circadian clock, finding a significant decrease in input resistance with age but no significant changes in spontaneous electrical activity or membrane potential. We propose this change contributes to observed behavioral and sleep changes in light-dark conditions. We also demonstrate a reduction in the daily plasticity of the architecture of the small ventral lateral neurons, likely underlying the reduction in circadian rhythmicity during aging. These results provide further insights into the effect of aging on circadian biology, demonstrating age-related changes in electrical activity in conjunction with the decline in behavioral outputs.

The HisCl1 histamine receptor acts in photoreceptors to synchronize Drosophila behavioral rhythms with light-dark cycles

In Drosophila, the clock that controls rest-activity rhythms synchronizes with light-dark cycles through either the blue-light sensitive cryptochrome (Cry) located in most clock neurons, or rhodopsin-expressing histaminergic photoreceptors. Here we show that, in the absence of Cry, each of the two histamine receptors Ort and HisCl1 contribute to entrain the clock whereas no entrainment occurs in the absence of the two receptors. In contrast to Ort, HisCl1 does not restore entrainment when expressed in the optic lobe interneurons. Indeed, HisCl1 is expressed in wild-type photoreceptors and entrainment is strongly impaired in flies with photoreceptors mutant for HisCl1. Rescuing HisCl1 expression in the Rh6-expressing photoreceptors restores entrainment but it does not in other photoreceptors, which send histaminergic inputs to Rh6-expressing photoreceptors. Our results thus show that Rh6-expressing neurons contribute to circadian entrainment as both photoreceptors and interneurons, recalling the dual function of melanopsin-expressing ganglion cells in the mammalian retina.

Cell-autonomous clock of astrocytes drives circadian behavior in mammals.

Circadian (~24-hour) rhythms depend on intracellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multicellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this pathway from gene to cell to circuit to behavior in mammals. We describe cell type-specific, functionally distinct TTFLs in neurons and astrocytes of the SCN and show that, in the absence of other cellular clocks, the cell-autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. Our results demonstrate that astrocytes can autonomously initiate and sustain complex mammalian behavior.

A Distinct Visual Pathway Mediates High-Intensity Light Adaptation of the Circadian Clock in Drosophila.

To provide organisms with a fitness advantage, circadian clocks have to react appropriately to changes in their environment. High-intensity (HI) light plays an essential role in the adaptation to hot summer days, which especially endanger insects of desiccation or prey visibility. Here, we show that solely increasing light intensity leads to an increased midday siesta in Drosophila behavior. Interestingly, this change is independent of the fly's circadian photoreceptor cryptochrome and is solely caused by a small visual organ, the Hofbauer-Buchner eyelets. Using receptor knock-downs, immunostaining, and recently developed calcium tools, we show that the eyelets activate key core clock neurons, namely the s-LNvs, at HI. This activation delays the decrease of PERIOD (PER) in the middle of the day and propagates to downstream target clock neurons that prolong the siesta. We show a new pathway for integrating light-intensity information into the clock network, suggesting new network properties and surprising parallels between Drosophila and the mammalian system.SIGNIFICANCE STATEMENT The ability of animals to adapt to their ever-changing environment plays an important role in their fitness. A key player in this adaptation is the circadian clock. For animals to predict the changes of day and night, they must constantly monitor, detect and incorporate changes in the environment. The appropriate incorporation and reaction to high-intensity (HI) light is of special importance for insects because they might suffer from desiccation during hot summer days. We show here that different photoreceptors have specialized functions to integrate low-intensity, medium-intensity, or HI light into the circadian system in Drosophila These results show surprising parallels to mammalian mechanisms, which also use different photoreceptor subtypes to respond to different light intensities.

Beyond spikes: Multiscale computational analysis of in vivo long-term recordings in the cockroach circadian clock.

The circadian clock of the nocturnal Madeira cockroach is located in the accessory medulla, a small nonretinotopic neuropil in the brain’s visual system. The clock comprises about 240 neurons that control rhythms in physiology and behavior such as sleep-wake cycles. The clock neurons contain an abundant number of partly colocalized neuropeptides, among them pigment-dispersing factor (PDF), the insects’ most important circadian coupling signal that controls sleep-wake rhythms. We performed long-term loose-patch clamp recordings under 12:12-hr light-dark cycles in the cockroach clock in vivo. A wide range of timescales, from milliseconds to seconds, were found in spike and field potential patterns. We developed a framework of wavelet transform–based methods to detect these multiscale electrical events. We analyzed frequencies and patterns of events with interesting dynamic features, such as mixed-mode oscillations reminiscent of sharp-wave ripples. Oscillations in the beta/gamma frequency range (20–40 Hz) were observed to rise at dawn, when PDF is released, peaking just before the onset of locomotor activity of the nocturnal cockroach. We expect that in vivo electrophysiological recordings combined with neuropeptide/antagonist applications and behavioral analysis will determine whether specific patterns of electrical activity recorded in the network of the cockroach circadian clock are causally related to neuropeptide-dependent control of behavior.

Astrocytes and Circadian Rhythms: An Emerging Astrocyte-Neuron Synergy in the Timekeeping System.

Animals have an internal timekeeping system to anticipate daily changes associated with the transition of day to night, which is deeply involved in the regulation and maintenance of behavioral and physiological processes. Prevailing knowledge associated the control of circadian clocks to a network of neurons in the central pacemaker, the suprachiasmatic nucleus (SCN), but astrocytes are rapidly emerging as key cellular contributors to the timekeeping system. However, how these glial cells impact the neuronal clock to modulate rhythmic neurobehavioral outputs just begin to be investigated. Astrocyte-neuron cocultures are an excellent exploratory method to further characterize the critical role of circadian communication between nerve cells, as well as to address the role of astrocytes as modulators and targets of neuronal rhythmic behaviors. Here, we describe a robust method to study astrocyte rhythmic interactions with neurons by coculturing them with primary neurons in physically separated layers. This simple coculture system provides hints on in vivo signaling processes. Moreover, it allows investigating cell-type specific effects separately as well as the identification of extracellular astrocytic or neuronal factors involved in rhythm generation in both cell types.

Circadian pacemaker neurons of the Madeira cockroach are inhibited and activated by GABAA and GABAB receptors.

GABA is the most abundant neurotransmitter in the circadian pacemaker circuits of mammals and insects. In the Madeira cockroach the accessory medulla (AME) in the brain's optic lobes is the circadian clock that orchestrates rest-activity rhythms in synchrony with light dark cycles. Three prominent GABAergic tracts connect the AME to termination sites of compound eye photoreceptors in the lamina and medulla. Parallel GABAergic light entrainment pathways were suggested to either advance or delay the clock for adjustment to changing photoperiods. In agreement with this hypothesis GABA activated or inhibited AME clock neurons, allowing for the distinction of three different GABA response types. Here, we examined which GABA receptors are responsible for these response types. We found that both ionotropic GABAA receptors and metabotropic GABAB receptors were expressed in AME clock cells. Via different signalling pathways, either one of them could account for all three GABA response types. The muscimol-dependently activated GABAA receptor formed a chloride channel, while the SKF 97541-dependently activated GABAB receptor signalled via G-proteins, apparently targeting potassium channels. Expression of chloride exporters or importers determined whether GABAA receptor activation hyper- or depolarized AME neurons. For GABAB receptor responses second messenger gated channels present in the clock cells appeared to decide about the polarity of the GABA response. In summary, circadian clock neurons co-expressed inhibitory and/or excitatory GABAA and GABAB receptors in various combinations, while cotransporter expression and the set of second messenger gated ion channels present allowed for distinct signalling in different clock neurons.

Allatostatin-C/AstC-R2 Is a Novel Pathway to Modulate the Circadian Activity Pattern in Drosophila

Seven neuropeptides are expressed within the Drosophila brain circadian network. Our previous mRNA profiling suggested that Allatostatin-C (AstC) is an eighth neuropeptide and specifically expressed in dorsal clock neurons (DN1s). Our results here show that AstC is, indeed, expressed in DN1s, where it oscillates. AstC is also expressed in two less well-characterized circadian neuronal clusters, the DN3s and lateral-posterior neurons (LPNs). Behavioral experiments indicate that clock-neuron-derived AstC is required to mediate evening locomotor activity under short (winter-like) and long (summer-like) photoperiods. The AstC-Receptor 2 (AstC-R2) is expressed in LNds, the clock neurons that drive evening locomotor activity, and AstC-R2 is required in these neurons to modulate the same short photoperiod evening phenotype. Exvivo calcium imaging indicates that AstC directly inhibits a single LNd. The results suggest that a novel AstC/AstC-R2 signaling pathway, from dorsal circadian neurons to an LNd, regulates the evening phase in Drosophila.

Bisphenol A Alters Bmal1, Per2, and Rev-Erba mRNA and Requires Bmal1 to Increase Neuropeptide Y Expression in Hypothalamic Neurons.

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, is considered an obesogen. However, its role in the hypothalamic control of energy balance remains largely unexplored. Because disruption of the circadian clock is tightly associated with metabolic consequences, we explored how BPA affects the components of the molecular circadian clock in the feeding-related neurons of the hypothalamus. In immortalized POMC and NPY/AgRP-expressing hypothalamic cell lines and primary culture, we describe how BPA significantly alters mRNA expression of circadian clock genes Bmal1,Per2, and Rev-Erbα. Furthermore, we use newly generated Bmal1-knockout (KO) hypothalamic cell lines to link the BPA-induced neuropeptide dysregulation to the molecular clock. Specifically, BPA increased Npy, Agrp, and Pomc mRNA expression in wild type hypothalamic cells, whereas the increase in Npy, but not Agrp or Pomc, was abolished in cell lines lacking BMAL1. In line with this increase, BPA led to increased BMAL1 binding to the Npy promotor, potentially increasing Npy transcription. In conclusion, we show that BPA-mediated dysregulation of the circadian molecular clock is linked to the deleterious effects of BPA on neuropeptide expression. Furthermore, we describe hypothalamic Bmal1-KO cell lines to study the role of BMAL1 in hypothalamic responses to metabolic, hormonal, and environmental factors.

Circadian and Sleep Circuits Ring Together

Prolonged wakefulness stimulates the homeostatic need to sleep, but transition to sleep also depends on the circadian time of day. However, links between circadian and homeostatic influences are not well understood. Guo etal. (2018) identify a Drosophila circuit connecting circadian clock neurons to sleep-promoting ring neurons in the ellipsoid body.

Clock-Generated Temporal Codes Determine Synaptic Plasticity to Control Sleep

Neurons use two main schemes to encode information: rate coding (frequency of firing) and temporal coding (timing or pattern of firing). While the importance of rate coding is well established, it remains controversial whether temporal codes alone are sufficient for controlling behavior. Moreover, the molecular mechanisms underlying the generation of specific temporal codes are enigmatic. Here, we show in Drosophila clock neurons that distinct temporal spike patterns, dissociated from changes in firing rate, encode time-dependent arousal and regulate sleep. From a large-scale genetic screen, we identifythe molecular pathways mediating the circadian-dependent changes in ionic flux and spike morphology that rhythmically modulate spike timing. Remarkably, the daytime spiking pattern alone is sufficient to drive plasticity in downstream arousal neurons, leading to increased firing of these cells. These findings demonstrate a causal role for temporal coding in behavior and define a form of synaptic plasticity triggered solely by temporal spike patterns.

A Wake-Promoting Circadian Output Circuit in Drosophila

Circadian clocks play conserved roles in gating sleep and wake states throughout the day-night cycle [1-5]. In the fruit fly Drosophila melanogaster, DN1p clock neurons have been reported to play both wake- and sleep-promoting roles [6-11], suggesting a complex coupling of DN1p neurons to downstream sleep and arousal centers. However, the circuit logic by which DN1p neurons modulate sleep remains poorly understood. Here, we show that DN1p neurons can be divided into two morphologically distinct subsets. Projections from one subset surround the pars intercerebralis, a previously defined circadian output region [12]. In contrast, the second subsetalso sends presynaptic termini to a visual processing center, the anterior optic tubercle (AOTU) [13]. Within the AOTU, we find that DN1p neurons inhibit a class of tubercular-bulbar (TuBu) neurons that act to promote consolidated sleep. These TuBu neurons in turn form synaptic connections with R neurons of the ellipsoid body, a region linked to visual feature detection, locomotion, spatial memory, and sleep homeostasis [14-17]. Our results define a second output arm from DN1p neurons and suggest a role for TuBu neurons as regulators of sleep drive.

Closely Related Fruit Fly Species Living at Different Latitudes Diverge in Their Circadian Clock Anatomy and Rhythmic Behavior.

Recently, we reported differences in the expression pattern of the blue light-sensitive flavoprotein cryptochrome (CRY) and the neuropeptide pigment-dispersing factor (PDF) in the neuronal clock network of high-latitude Drosophila species, belonging to the Drosophila subgenus ( virilis-repleta radiation), compared with cosmopolitan D. melanogaster flies, belonging to the Sophophora subgenus. Alterations in rhythmic patterns of activity due to these differences might have adaptive significance for colonizing high-latitude habitats and, hence, adjusting to long photoperiods. Here, we show that these differing CRY/PDF expression patterns are only present in those species of the virilis-repleta radiation that colonized high latitudes. The cosmopolitan species D. mercatorum and D. hydei have a D. melanogaster-like clock network and behavior despite belonging to the virilis-repleta radiation. Similarly, 2 species of the holotropical Zaprionus genus, more closely related to the Drosophila subgenus than to the Sophophora subgenus, retain a D. melanogaster-like clock network and rhythmic behavior. We therefore suggest that the D. melanogaster-like clock network is the "ancestral fly clock phenotype" and that alterations in the CRY/PDF clock neurochemistry have allowed some species of the virilis-repleta radiation to colonize high-latitude environments.

The CCHamide1 Neuropeptide Expressed in the Anterior Dorsal Neuron 1 Conveys a Circadian Signal to the Ventral Lateral Neurons in Drosophila melanogaster.

The fruit fly Drosophila melanogaster possesses approximately 150 brain clock neurons that control circadian behavioral rhythms. Even though individual clock neurons have self-sustaining oscillators, they interact and synchronize with each other through a network. However, little is known regarding the factors responsible for these network interactions. In this study, we investigated the role of CCHamide1 (CCHa1), a neuropeptide expressed in the anterior dorsal neuron 1 (DN1a), in intercellular communication of the clock neurons. We observed that CCHa1 connects the DN1a clock neurons to the ventral lateral clock neurons (LNv) via the CCHa1 receptor, which is a homolog of the gastrin-releasing peptide receptor playing a role in circadian intercellular communications in mammals. CCHa1 knockout or knockdown flies have a generally low activity level with a special reduction of morning activity. In addition, they exhibit advanced morning activity under light-dark cycles and delayed activity under constant dark conditions, which correlates with an advance/delay of PAR domain Protein 1 (PDP1) oscillations in the small-LNv (s-LNv) neurons that control morning activity. The terminals of the s-LNv neurons show rather high levels of Pigment-dispersing factor (PDF) in the evening, when PDF is low in control flies, suggesting that the knockdown of CCHa1 leads to increased PDF release; PDF signals the other clock neurons and evidently increases the amplitude of their PDP1 cycling. A previous study showed that high-amplitude PDP1 cycling increases the siesta of the flies, and indeed, CCHa1 knockout or knockdown flies exhibit a longer siesta than control flies. The DN1a neurons are known to be receptive to PDF signaling from the s-LNv neurons; thus, our results suggest that the DN1a and s-LNv clock neurons are reciprocally coupled via the neuropeptides CCHa1 and PDF, and this interaction fine-tunes the timing of activity and sleep.

Molecular and circuit mechanisms mediating circadian clock output in the Drosophila brain.

A central question in the circadian biology field concerns the mechanisms that translate ~24-hr oscillations of the molecular clock into overt rhythms. Drosophila melanogaster is a powerful system that provided the first understanding of how molecular clocks are generated and is now illuminating the neural basis of circadian behavior. The identity of ~150 clock neurons in the Drosophila brain and their roles in shaping circadian rhythms of locomotor activity have been described before. This review summarizes mechanisms that transmit time-of-day signals from the clock, within the clock network as well as downstream of it. We also discuss the identification of functional multisynaptic circuits between clock neurons and output neurons that regulate locomotor activity.

Cryptochrome Interacts With Actin and Enhances Eye-Mediated Light Sensitivity of the Circadian Clock in Drosophila melanogaster.

Cryptochromes (CRYs) are a class of flavoproteins that sense blue light. In animals, CRYs are expressed in the eyes and in the clock neurons that control sleep/wake cycles and are implied in the generation and/or entrainment of circadian rhythmicity. Moreover, CRYs are sensing magnetic fields in insects as well as in humans. Here, we show that in the fruit fly Drosophila melanogaster CRY plays a light-independent role as “assembling” protein in the rhabdomeres of the compound eyes. CRY interacts with actin and appears to increase light sensitivity of the eyes by keeping the “signalplex” of the phototransduction cascade close to the membrane. By this way, CRY also enhances light-responses of the circadian clock.

Iron Deficiency Reduces Synapse Formation in the Drosophila Clock Circuit

Iron serves as a critical cofactor for proteins involved in a host of biological processes. In most animals, dietary iron is absorbed in enterocytes and then disseminated for use in other tissues in the body. The brain is particularly dependent on iron. Altered iron status correlates with disorders ranging from cognitive dysfunction to disruptions in circadian activity. The exact role iron plays in producing these neurological defects, however, remains unclear. Invertebrates provide an attractive model to study the effects of iron on neuronal development since many of the genes involved in iron metabolism are conserved, and the organisms are amenable to genetic and cytological techniques. We have examined synapse growth specifically under conditions of iron deficiency in the Drosophila circadian clock circuit. We show that projections of the small ventrolateral clock neurons to the protocerebrum of the adult Drosophila brain are significantly reduced upon chelation of iron from the diet. This growth defect persists even when iron is restored to the diet. Genetic neuronal knockdown of ferritin 1 or ferritin 2, critical components of iron storage and transport, does not affect synapse growth in these cells. Together, these data indicate that dietary iron is necessary for central brain synapse formation in the fly and further validate the use of this model to study the function of iron homeostasis on brain development.

Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster.

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.